Drug delivery system for administration of poorly water soluble pharmaceutically active substances

ABSTRACT

This invention relates to a drug delivery system for administration of poorly water soluble pharmaceutically active substance, a pharmaceutical composition comprising such a drug delivery system, and a method for the preparation of such a drug delivery system. The invention also relates to a method for controlling the particle size and/or particle shape and/or particle size distribution in such a drug delivery system, and to a method for increasing the drug loading capacity of the particles. Furthermore the invention also relates to the use of such a drug delivery system for the preparation of a medicament for the treatment of cancer.

This application is a continuation of U.S. application Ser. No.12/809,252 filed on Sep. 15, 2010, which is a national phase applicationof International Application No. PCT/SE2008/051515 filed on Dec. 18,2008 and published in the English language, which claims priority toInternational Application No. PCT/SE2007/001127 filed on Dec. 19, 2007.

FIELD OF THE INVENTION

This invention relates to a drug delivery system for administration ofpoorly water soluble pharmaceutically active substances, apharmaceutical composition comprising such a drug delivery system, and amethod for the preparation of such a drug delivery system. The inventionalso relates to a method for controlling the particle size and/orparticle shape and/or particle size distribution in such a drug deliverysystem, and to a method for increasing the drug loading capacity of theparticles. Furthermore the invention also relates to the use of such adrug delivery system for the preparation of a medicament for thetreatment of cancer.

BACKGROUND OF THE INVENTION

There is a critical need in the pharmaceutical and other relatedindustries to formulate industrially useful water-insoluble or poorlywater soluble substances into formulations for oral, injectable,inhalation, ophthalmic, and other routes of delivery. Industriallyuseful water insoluble or poorly water soluble substances include waterinsoluble or poorly water soluble biologically useful compounds, imagingagents, pharmaceutically useful compounds and in particular waterinsoluble and poorly water soluble drugs for human and veterinarymedicine.

No limitation is imposed on the kind of water-insoluble or poorly watersoluble substances for use in the present invention. Examples includeantipyretics, anti-inflammatories, analgesics, ataractics, sedatives,antitumor agents, antimicrobials, antibiotics, antilipemics,antitussives/expectorants, muscle relaxants, antiepileptics, antiulcers,antidepressants, antiallergics, cardiotonics, antiarrhythmics,vasodilators, hypotensors/diuretics, diabetes therapeutics,tuberculostatics, antirheumatics, steroids, narcotic antagonists,hormones, fat-soluble vitamins, anticoagulants, ischemic diseasetherapeutics, immune disease therapeutics, Alzheimer's diseasetherapeutics, osteoporosis therapeutics, angiopoiesis therapeutics,retinosis therapeutics, retinal vein occlusion therapeutics, seniledisciform macular degeneration, cerebrovascular spasm therapeutics,cerebral thrombosis therapeutics, cerebral infarction therapeutics,cerebral occlusion therapeutics, intracerebral hemorrhage therapeutics,subarachnoid hemorrhage therapeutics, hypertensive encephalopathytherapeutics, transient cerebral ischemic attack therapeutics,multi-infarct dementia therapeutics, arterial sclerosis therapeutics,Huntington's disease therapeutics, brain tissue disorder therapeutics,optic neuropathy therapeutics, glaucoma therapeutics, ocularhypertension therapeutics, retinal detachment therapeutics, arthritistherapeutics, antisepsis drugs, antiseptic shock drugs, antiasthmadrugs, pollakiuria/incontinentia therapeutics, atopic rhinitistherapeutics, allergic rhinitis therapeutics, cosmetic compositions,agrichemical compositions, insecticides, bactericides, herbicides,beverage or food compositions, immunosuppressants and animal drugcompositions.

The fact that only water soluble substances can be administratedintravenously considerably impoverishes the assortment of organicmolecules that can be used as antineoplastic drugs, as many if not mostof these are poorly water soluble.

Incorporation of polar functions into such substances does not solvethis problem because the changes of the structure lead to loss of therelevant pharmacological properties of the drugs.

Development of drug delivery systems which could enable dissolvation ofpoorly soluble compounds in aqueous solutions would be hugelyinstrumental in the efforts of realizing the anticancer potential of avast number of substances, and would provide for creation of novelgenerations of drugs.

Paclitaxel and docetaxel belong to the taxane class of anticancer drugsbecause they or their precursors are produced by the plants of the genusTaxus (yews). Paclitaxel is still produced by isolation from naturalsources while docetaxel, a semi-synthetic analogue of paclitaxel, issynthesized from 10-deacetyl baccatin. Paclitaxel differs from docetaxelby an acetylated hydroxyl function at position 10 and a benzoyl moietyinstead of tert-butyl on the phenylpropionate side chain. The mechanismof action of taxanes is based on their ability to bind to the β subunitof tubulin which interferes with the depolymerization of microtubules,thereby damaging dividing cells. This specificity of action is widelyused in oncology to treat different solid tumors, especially ovarian,lung, breast, bladder, head and neck cancer. Paclitaxel and docetaxelhave poor oral bioavailability and therefore intravenous (i.v.) infusionis the only way of administration. Scarce water solubility also makes itimpossible to use aqueous solutions of these taxanes. Several deliveryvehicles have been applied to solve this problem.

TAXOL® is based on the ability of CREMOPHOR® EL, a polyethoxylatedcastor oil, to dissolve paclitaxel in the weight-to-weight (w/w) ratio87:1. It is chronologically the first commercial taxane formulationwhich has opened the era of taxane use in oncology. However it was laterfound that CREMOPHOR® is the cause of hypersensitivity reactions duringTAXOL® infusion and for minimization of the incidence and severity ofthese reactions a premedication with histamine blockers andglucocorticoids as well as continuous infusion schedules became standardpractice.

In a second delivery system called TAXOTERE®, Polysorbate 80 (knownunder the trademark TWEEN®80), a derivative of polyethoxylated sorbitoland oleic acid, plays the role of vehicle. In this case the w/w ratio is24:1. Like CREMOPHOR® EL, Polysorbate 80 is a non-ionic detergent buildof polyethoxy chains and can also induce hypersensitivity reactions.

ABRAXANE®, a third delivery system, consists of paclitaxel nanoparticlesstabilized by human serum albumin in the w/w ratio 9:1 with the meandiameter of nanoparticles being 130 nm. The absence of non-ionicsurfactants simplifies the treatment as no premedication is necessaryand the infusion time is shortened. On the other hand the ABRAXANE®formulation is less potent than TAXOL® because ABRAXANE® nanoparticleslike other particles with the size more than 100 nm are a substrate forreticuloendothelial system. Another disadvantage of this drug deliveryvehicle is that human serum albumin isolated from donor blood is used,which always carries a small but definite risk of transmission of viraldiseases.

Finally, it has been found that paclitaxel and docetaxel can bedissolved in aqueous solutions of water-soluble derivatives of retinoicacid acting as anionic surfactants. The uniqueness of the structure ofthese derivatives enables them to dissolve paclitaxel and docetaxel inthe surprisingly low w/w ratio of 0.5:1. Ixabepilone, (epothilone Banalog) is very similar to taxanes in terms of mode of action andsolubility in aqueous solutions. It is indicated for the treatment ofmetastatic or locally advanced breast cancer. Formulation of ixabepilonefor IV administration, Ixempra, developed by BMS, like Taxol, is basedon cremophor EL and therefore a premedication and prolonged infusion forthe reducing of hypersensitivity reactions is required.

Etoposide, analog of toxin podophyllotoxin, is topoisomerase IIinhibitor and is used for treatment of Ewing's sarcoma, lung cancer,testicular cancer, lymphoma and non-lymphocytic leukemia. Etoposideformulations for IV administration are based on PEG-derivatives such asPolysorbate 80 (TWEEN 80) or Macrogol 300 in order to solubilize thescarce water soluble active pharmaceutical ingredient.

Retinoids comprise a family of polyisoprenoids which includes vitamin A(retinol) and its natural (retinoic acid) and synthetic analogs(fenretinide, etretinate, tazarotene, bexarotene, adapalene). Thesecompounds show a broad spectrum of biological activity includingparticipation in control of cell proliferation, cell differentiation andembryonic development which enables to use retinoids as antineoplasticagents for treatment of different types of cancer such as leukemia,lymphoma, Kaposi's sarcoma, lung cancer and breast cancer. Thesecompounds are also used for treatment of different skin disease likepsoriasis, acne, and sun damaged skin. Retinoids are usually highlylipophilic compounds and their usage in form of aqueous solution demandsapplication of some delivery system. However so far there are no anycommercially developed water-soluble formulations of retinoids and theyare available exclusively for oral administration.

Ciclosporin, sirolimus, tacrolimus, and everolimus areimmunosuppressants which are scarcely water soluble. Bioavailability ofthe drugs at oral administration is only about 20%. Commerciallyavailable formulations of these immunosuppressants are based solely onthe use of polyoxyethylated castor oil, which causes hypersensitivityreactions when intravenously administered.

Ciclosporin, cyclosporine, or cyclosporin, is an immunosuppressant drugwidely used in post-allogeneic organ transplant to reduce the activityof a patient's immune system and so the risk of organ rejection. It hasbeen studied in transplants of skin, heart, kidney, liver, lung,pancreas, bone marrow and small intestine. Initially isolated from aNorwegian soil sample, Ciclosporin A, the main form of the drug, is acyclic nonribosomal peptide of 11 amino acids (an undecapeptide)produced by the fungus Tolypocladium inflatum Gams, and contains D-aminoacids, which are rarely encountered in nature.

The search for and development of new drug delivery systems hasincreased with the realization of the fact that drugs in too highconcentrations are toxic and—in best case—inactive in too lowconcentrations; however, exposing a cell to a too low concentrations ofdrugs often activates mechanisms of resistance to the drug. The range ofconcentrations where the drug elicits the desired response with lessside-effects is known as “the therapeutic window”.

Prolonged infusions have been proven to reduce the toxicity ofanticancer agents but this mode of administration is significantly morecomplicated from a practical point of view.

It has been found that slow drug release can be achieved by using drugsthat are bound or encapsulated in nanoparticles of different kind. Theseparticles can circulate in blood for several days playing the role of“depots”. The drug release occurs by diffusion of the encapsulated drugsor by erosion and decomposition of the particles. The most popular typesof nanoparticles in this field of research are micelles and liposomes asthe formation of such nanoparticles is a quite simple entropy drivenprocess, i.e. they emerge spontaneously and their properties areprogrammed by conditions of the formation. The size of particles used inthese delivery systems is within the range of 8 to 200 nm and evenhigher.

However, with the increase of the size, a particle becomes “visible” tothe reticulo-endothelial system, a part of the immune system consistingof the phagocytic cells located in reticular connective tissue of lymphnodes, liver and spleen. The extent of reticulo-endothelial systemclearance increases with the size of the particles, significantlyreducing the total amount of the drug in the blood flow.

Another intriguing challenge in the field of drug delivery is thetargeting of drugs to effect compartments which could increase thetherapeutic effectiveness up to ultimate levels. Nanoparticles have beenfound very useful in this regard. Solid tumors differ pathoanatomicallyfrom healthy tissues by an extensive angiogenesis, as well as ahyperpermeable and defective vasculature architecture. In other wordsthe size of the tumour capillaries is larger, making it potentiallypossible to significantly increase the passive transport ofnanoparticles loaded with cytotoxic cargo to the tumour in comparison toa healthy endothelium.

US 2004048923 describes a group of retinoids including among numerousothers the sodium salt of N-(all-trans-retinoyl)-L-cysteic acid methylester and the sodium salt of N-(13-cis-retinoyI)-L-cysteic acid methylester. It is stated that the substances make it possible to manufacturenew micelle formulations of poorly soluble pharmaceutical compounds likepaclitaxel and docetaxel.

WO 02092600 relates to a method for preparing a water-solubleformulation of paclitaxel, comprising the steps of dissolving paclitaxelin a first solvent, dissolving a compound chosen amongN-(all-trans-Retinoyl)-L-cysteic acid, N-(13-cis-Retinoyl)-L-cysteicacid, N-(all-trans-Retinoyl)-L-homocysteic acid,N-(13-cis-Retinoyl)-L-homocysteic acid,N-(all-trans-Retinoyl)-L-cysteinesulfinic acid, andN-(13-cis-Retinoyl)-L-cysteinesulfinic acid in a second solvent, mixingthe aliquots of the resulting solutions of paclitaxel and the saidcompound in a desired molar ratio, and evaporating the resulting mixtureto dryness.

Although the poor solubility of the pharmaceutical compounds may suggestthat they are in particular form, US 2004048923 and WO 02092600 are bothcompletely silent regarding the size and the morphology of theparticles. There is in particular no indication or suggestion that theyshould be in amorphous state, or even that they could exist in such astate. Little less is any way of providing particles in such a statedisclosed. As well known to those skilled in the polymorphism, includingpossible amorphism, is basically unpredictable for organic substances.

SHORT SUMMARY OF THE INVENTION

The creation of a new drug delivery system with controlled or in advanceprogrammed drug release mimicking prolonged administrations would begreatly desired.

One object of the present invention is to provide such a drug deliverysystem.

Thus, one aspect of the invention relates to a drug delivery system foradministration of at least one pharmaceutically active substance havinga solubility per se in water of less than about 100 μg/ml, saidsubstance being in particulate form with an effective average particlesize of less than about 100 nm, wherein the substance particles areessentially amorphous; the substance particles are entrapped innanoparticles formed of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or a combination thereof; and theweight-to-weight ratio of said sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or combination thereof, to saidsubstance is in the range from 0.5:1 to 20:1.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in closer detail in thefollowing description, examples and attached drawings, in which

FIG. 1 shows the dependence of the particle size on the w/w ratio ofsodium salt of methyl ester of N-all-trans-retinoyl cysteic acid topaclitaxel at different paclitaxel concentrations in an aqueous solutionof sodium chloride at a concentration of 9 mg/ml.

FIG. 2 shows the dependence of the size of particles formed by sodiumsalt of methyl ester of N-13-cis-retinoyl cysteic acid and docetaxel(w/w ratio 1:1) on the concentration of sodium chloride at differentdocetaxel concentrations.

FIG. 3 shows the dependence of the size of particles formed by sodiumsalt of methyl ester of N-all-trans-retinoyl cysteic acid and paclitaxel(w/w ratio of paclitaxel:methyl ester of N-all-trans-retinoyl cysteicacid is 1:2) on the concentration of calcium chloride in an aqueoussolution of sodium chloride at a concentration of 9 mg/ml.

FIGS. 4 and 5 show the time course of particle size and Z-potential of aformulation obtained by reconstitution of a freeze dried mixture ofpaclitaxel, sodium salt of methyl ester of N-all-trans-retinoyl cysteicacid and sodium salt of methyl ester of N-13-cis-retinoyl cysteic acidin w/w/w ratio 1:0.75:0.75 in an aqueous solution of sodium chloride (9mg/ml), calcium chloride (2 mmol/l) and magnesium chloride (1 mmol/l).

FIGS. 6 and 7 show the time course of particle size and Z-potential of aformulation obtained by reconstitution of a freeze dried mixture ofdocetaxel and sodium salt of methyl ester of N-all-trans-retinoylcysteic acid in w/w ratio 1:2 in an aqueous solution of sodium chloride(9 mg/ml) and calcium chloride (3 mmol/l)

FIG. 8 shows a comparative evaluation of the cytotoxicity of theformulations formed by docetaxel-sodium salt of methyl ester ofN-all-trans-retinoyl cysteic acid-sodium salt of methyl ester ofN-13-cis-retinoyl cysteic acid mixture (w/w/w=1:1:1) in cultures ofhuman ovary adenocarcinoma SKOV3 cell line.

FIG. 9 shows a comparative evaluation of the cytotoxicity of theformulations formed by paclitaxel-sodium salt of methyl ester ofN-all-trans-retinoyl cysteic acid-sodium salt of methyl ester ofN-13-cis-retinoyl cysteic acid mixture (w/w/w=1:0.75:0.75) in culturesof human ovary adenocarcinoma SKOV3 Cell Line.

FIG. 10 shows the dependence of the particle size on the w/w ratio ofsodium salt of methyl ester of N-all-trans-retinoyl cysteic acid toCiclosporin A at different Ci-closporin A concentrations in an aqueoussolution of sodium chloride at a concentration of 9 mg/ml.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Before the present invention is disclosed and described, it is to beunderstood that this invention is not limited to the particularconfigurations, process steps, and materials disclosed herein as suchconfigurations, process steps, and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise.

In this specification, unless otherwise stated, the term “about”modifying the quantity of an ingredient in the drug delivery systems orcompositions of the invention or employed in the methods of theinvention refers to variation in the numerical quantity that can occur,for example, through typical measuring and liquid handling proceduresused for making concentrates or use solutions in the real world; throughinadvertent error in these procedures; through differences in themanufacture, source, or purity of the ingredients employed to make thedrug delivery systems or compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

In this specification, unless otherwise stated, the term “drug deliverysystem” refers to a formulation or device that delivers therapeuticagent(s) to desired body location(s) and/or provides timely release oftherapeutic agent(s).

In this specification, unless otherwise stated, the term “particle size”refers to the Z-average diameter as measured by dynamic light scatteringwith the use of red laser with a wavelength of 633 nm. By “an effectiveaverage particle size of less than about 100 nm” it is meant that atleast 90% of the particles have a size of less than about 100 nm whenmeasured by the above-noted technique.

In this specification, unless otherwise stated, the term “nanoparticle”refers to a microscopic particle whose size is measured in nanometres.Nanoparticles of the invention typically range from about 1 to about 999nm in diameter, and can include an entrapped, encapsulated, or enclosedbiologically active molecule.

In this specification, unless otherwise stated, the term “solubility” ofa substance refers to the ability of that substance to be dissolved in aspecified solvent at about room temperature, by which is meant frombetween about 15° C. to about 38° C.

In this specification, unless otherwise stated, the term “amorphous” isintended to indicate a solid structure that is either non-crystalline orconsists of very small crystals having a particle size of about 10 nm orless.

In this specification, unless otherwise stated, the term “cytotoxiccompound” refers to a compound that has the ability of arresting thegrowth of, or killing, cells.

In this specification, unless otherwise stated, the term “cytostaticcompound” refers to a compound that has the ability of bringing cells,although not necessarily lysed or killed, into a permanentnon-proliferative state.

In this specification, unless otherwise stated, the term“immunosuppressant” refers to a compound that has the ability ofinhibiting the activity of the immune system, in particular forpreventing rejection of a transplant organ and in disorders where thebody's immune system attacks its own tissues

In this specification, unless otherwise stated, the term “derivative”refers to a compound formed from the original structure either directly,by chemical reaction of the original structure, or by a “modification”which is a partial substitution of the original structure, or by designand de novo synthesis. Derivatives may be synthetic, or may be metabolicproducts of a cell or an in vitro enzymatic reaction.

In one embodiment the substance particles in the inventive drug deliverysystem have an effective average particle size of less than about 50 nm.

In another embodiment the substance particles in the inventive drugdelivery system have an effective average particle size in the range ofabout 5-50 nm.

In yet another embodiment the substance particles in the inventive drugdelivery system have an effective average particle size in the range ofabout 8-30 nm.

In one embodiment of the present invention the weight-to-weight ratio ofthe sodium salt of methyl ester of N-all-trans-retinoyl cysteic acid,sodium salt of methyl ester of N-13-cis-retinoyl cysteic acid, orcombination thereof, to the pharmaceutically active substance is in therange from about 1:1 to about to 10:1.

In one embodiment of the present invention the pharmaceutically activesubstance is a cytotoxic or a cytostatic compound; in one aspect of thisembodiment the cytotoxic or cytostatic compound is bischloronitrosourea(Carmustine); in another aspect of this embodiment the cytotoxic orcytostatic compound is etoposide; in yet another aspect of thisembodiment the cytotoxic or cytostatic compound is a taxane, and in amore specific aspect the taxane is chosen among paclitaxel, docetaxel,and derivatives thereof. In another specific aspect of said embodimentthe invention relates to such a drug delivery system for use intreatment of cancer.

In one embodiment of the present invention the pharmaceutically activesubstance is an immunosuppressant; in one aspect of this embodiment theimmunosuppressant is chosen among ciclosporin, sirolimus, tacrolimus andderivatives thereof. In another aspect of said embodiment the inventionrelates to such a drug delivery system for use in post-allogeneic organtransplant.

Another embodiment of the invention relates to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and a drugdelivery system of this kind.

In one aspect of this embodiment the pharmaceutically active substanceis a cytotoxic or a cytostatic compound; in one aspect of thisembodiment the cytotoxic or cytostatic compound is bischloronitrosourea(Carmustine); in another aspect of this embodiment the cytotoxic orcytostatic compound is etoposide; in yet another aspect of thisembodiment the compound is a taxane, which may be chosen amongpaclitaxel, docetaxel, and derivatives thereof; in another aspect ofthis embodiment of the present invention the pharmaceutically activesubstance is an immunosuppressant; in one aspect of this embodiment theimmunosuppressant is chosen among ciclosporin, sirolimus, tacrolimus andderivatives thereof. In one aspect of this embodiment the pharmaceuticalcomposition may be provided in the form of an aqueous solution, a gel, acream, an ointment, a tablet, a capsule, or a softgel.

A further embodiment of the invention relates to the use of a sodiumsalt of the methyl ester of N-all-trans-retinoyl cysteic acid, a sodiumsalt of the methyl ester of N-13-cis-retinoyl cysteic acid, or acombination thereof, in the preparation of such a drug delivery system.

Yet another embodiment of the invention relates to a method for thepreparation of a drug delivery system comprising nanoparticles formed ofa sodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid,a sodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid, ora combination thereof, and at least one pharmaceutically activesubstance having a solubility per se in water of less than about 100μg/ml, wherein said substance is provided in the form of essentiallyamorphous particles with an effective average particle size of less thanabout 100 nm; the size of said nanoparticles is controlled to have aneffective average particle size of less than about 100 nm by adjustingthe weight-to-weight ratio of said sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or combination thereof, to saidsubstance to be in the range from about 0.5:1 to about 20:1. The presentinvention also provides a drug delivery system obtainable by this methodas well as a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and such a drug delivery system.

A yet further embodiment of the invention relates to a method forcontrolling the particle size and/or particle shape and/or particle sizedistribution of nanoparticles formed of a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid, a sodium salt of the methylester of N-13-cis-retinoyl cysteic acid, or a combination thereof, andat least one pharmaceutically active substance having a solubility perse in water of less than about 100 μg/ml in a process for thepreparation of a drug delivery system, wherein said substance isprovided in the form of essentially amorphous particles with aneffective average particle size of less than about 100 nm; the particlesize and/or particle shape and/or particle size distribution of saidnanoparticles is controlled by adjusting the weight-to-weight ratio ofsaid sodium salt of the methyl ester of N-all-trans-retinoyl cysteicacid, sodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid,or combination thereof, to said substance to be in the range from about0.5:1 to about 20:1. In one aspect of this embodiment the size of thenanoparticles is controlled to be in the range of about 10-100 nm.

Yet another embodiment of the invention relates to a method forcontrolling the particle size of nanoparticles formed of a sodium saltof the methyl ester of N-all-trans-retinoyl cysteic acid, a sodium saltof the methyl ester of N-13-cis-retinoyl cysteic acid, or a combinationthereof, and at least one pharmaceutically active substance having asolubility per se in water of less than about 100 μg/ml in a process forthe preparation of a drug delivery system, wherein said substance isprovided in the form of essentially amorphous particles with aneffective average particle size of less than about 100 nm; saidessentially amorphous particles are submitted into and/or produced in anaqueous solution containing at least one ionized salt, said aqueoussolution having an ionic strength I; and the particle size of thenanoparticles is increased by increasing I or decreased by decreasing I.

In one aspect of this embodiment the pharmaceutically active substanceis a taxane and said at least one ionized salt is sodium chloride. Thisis useful for the production of i.v. infusion solutions as sodium andchloride ions are the most abundant ions in the human body and also inthe bodies of many animals.

In another aspect of this embodiment of this embodiment the ionized saltcomprises polyvalent cations, such as, for instance, double valencedcations. Such cations do not only increase the ionic strength of thesolvent in general, thereby increasing the particle size, but alsostabilize the particles formed.

The use of taxane-containing particles having a size within the range ofabout 10-100 nm significantly improves the therapeutic efficacy of theseanti-cancer compounds by extension a blood circulation of the drugs,lowering their reticuloendothelial clearance and selective penetrationof defective vasculature. Besides the advantages of the use of taxanesin the form of such nanoparticles in vivo, i.e. slow drug release andincreased permeability of tumour vasculature, it has also been foundthat the activity of taxane formulations containing such nanoparticlesis more expressed in vitro in different solid tumour cell lines.Moreover the cytotoxicity of these formulations dramatically depends onthe particle size.

Another embodiment of the invention relates to a method for increasingthe drug loading capacity of nanoparticles formed of a sodium salt ofthe methyl ester of N-all-translretinoyl cysteic acid, a sodium salt ofthe methyl ester of N-13-cis-retinoyl cyteic acid, or a combinationthereof, and at least one pharmaceutically active substance having asolubility per se in water of less than about 100 μg/ml in a process forthe preparation of a drug delivery system by providing said substance inthe form of essentially amorphous particles with an effective averageparticle size of less than about 100 nm; and adjusting theweight-to-weight ratio of said sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or combination thereof, to saidsubstance to be in the range from about 0.5:1 to about 20:1.

In each one of said inventive methods the pharmaceutically activesubstance may be provided in the form of essentially amorphous particleswith an effective average particle size of less than about 100 nm by wayof a method comprising the steps of: dissolving said substance in asuitable organic solvent to provide an organic solution of saidsubstance; adding about 0.01-3 molar equivalents of a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid, a sodium salt of themethyl ester of N-13-cis-retinoyl cysteic acid, or a combinationthereof, to said organic solution; and evaporating said organic solventfrom said organic solution to provide a residue which comprises thepharmaceutically active substance in the form of essentially amorphousparticles. In one embodiment of this method about 0.1-1 molarequivalents of a sodium salt of the methyl ester of N-all-trans-retinoylcysteic acid, a sodium salt of the methyl ester of N-13-cis-retinoylcysteic acid, or a combination thereof, is added to the organicsolution.

The proposed method is based on the ability of a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid, as well as a sodiumsalt of the methyl ester of N-13-cis-retinoyl cysteic acid to preventcrystallization of pharmaceutically active substance such as, forinstance, taxanes.

During the evaporation of the organic solvent the sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid, sodium salt of themethyl ester of N-13-cis-retinoyl cysteic acid, or combination thereof,co-crystallize with the pharmaceutically active substance, forming afilm. Water added to this film dissolves the sodium salt of the methylester of N-all-trans-retinoyl cysteic acid, sodium salt of the methylester of N-13-cis-retinoyl cysteic acid, or combination thereof, andprovides the pharmaceutically active substance in a highly amorphousform with tremendously increased surface area.

The thus obtained solution of essentially amorphous particles of thepharmaceutically active substance can be used directly without isolationor purification for infusions or for the manufacturing of freeze driedproducts for future reconstitutions.

Alternatively, the essentially amorphous particles of thepharmaceutically active substance can be provided in dry form by way of,for instance, evaporation, and then later on be dissolved in an aqueoussolution comprising about 0.01-50 molar equivalents of said sodium saltof the methyl ester of N-all-trans-retinoyl cysteic acid, sodium salt ofthe methyl ester of N-13-cis-retinoyl cysteic acid, or combinationthereof. In one embodiment said active substance particles may bedissolved in such a solution comprising about 0.1-5 molar equivalents ofa sodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid,a sodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid, orcombination thereof. The essentially amorphous particles are possible todissolve in a solution of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or combination thereof, within a coupleof minutes.

In another alternative, a solution of the pharmaceutically activesubstance in an organic solvent is added to an aqueous solution of asodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid, asodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid, or acombination thereof, whereafter the organic solvent is evaporated,leaving an aqueous solution comprising the pharmaceutically activesubstance in an amorphous form.

This method can be optimized and simplified by arranging influx oforganic solution of the pharmaceutically active substance into anevaporation flask containing an aqueous solution of a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid, a sodium salt of themethyl ester of N-13-cis-retinoyl cysteic acid, or a combinationthereof, simultaneously with evaporation.

The flow-rate of the organic solution, the internal pressure in theevaporation system as well as the evaporation temperature may be chosenin such a way that concentration of organic solution does not exceed15%.

The organic solvent used in the process for providing thepharmaceutically active substance in the form of essentially amorphousparticles may be an alcohol such as, for instance, methanol or ethanol.The use of methanol which has lower boiling point instead of ethanolsimplifies the evaporation of the alcohol-water mixtures.

However, as residues of organic solvent may be less appropriate fordirect in vivo application, the organic solutions of the essentiallyamorphous particles of pharmaceutically active substance may, forinstance, be freeze-dried to remove the organic solvent, leaving theessentially amorphous particles of pharmaceutically active substance ina convenient powder form for storage and preparation of newformulations.

According to other embodiments of the present invention there is alsoprovided:

-   -   the use of the inventive drug delivery system for the        preparation of a medicament for the treatment of cancer, and to        a method for the treatment of cancer wherein the inventive drug        delivery system is administered in a therapeutically effective        amount to a patient in need of such treatment; and    -   the use of the inventive pharmaceutical composition for the        preparation of a medicament for the treatment of cancer, and to        a method for the treatment of cancer, wherein the inventive        pharmaceutical composition is administered in a therapeutically        effective amount to a patient in need of such treatment.    -   the use of the inventive drug delivery system for the        preparation of a medicament for use in post-allogeneic organ        transplant, and to a method for post-allogeneic organ transplant        wherein the inventive drug delivery system is administered in a        therapeutcally effective amount to a patient in need of such        treatment; and    -   the use of the inventive pharmaceutical composition for the        preparation of a medicament for use in post-allogeneic organ        transplant, and to a method for post-allogeneic organ        transplant, wherein the inventive pharmaceutical composition is        administered in a therapeutically effective amount to a patient        in need of such treatment.

The water soluble taxane formulations obtained with the use of a sodiumsalt of the methyl ester of N-all-trans-retinoyl cysteic acid, a sodiumsalt of the methyl ester of N-13-cis-retinoyl cysteic acid, or acombination thereof, are stable for several hours in the broad intervalof conditions of formation of these formulations.

Thus, the present invention makes it possible to provide aqueoussolutions of taxanes with otherwise poor water solubility, likepaclitaxel and docetaxel, for infusion without any use of non-ionicsurfactants. This significantly reduces hypersensitivity reactionagainst the infusion solutions, shortens the infusion time, and obviatesthe need of premedication of patients against such hypersensitivity.

The invention will be illustrated in closer detail in the followingnon-limiting examples.

EXAMPLES Materials and Methods

Formulations of active pharmaceutical ingredients with a sodium salt ofthe methyl ester of N-all-trans-retinoyl cysteic acid, a sodium salt ofthe methyl ester of N-13-cis-retinoyl cysteic acid, or a combinationthereof, were prepared by reconstitution of either freshly evaporated orfreeze dried residues of an active ingredient with the retinoyl cysteicacid derivatives by a specified solution for reconstitution.

Paclitaxel, Ciclosporine A and all-trans-retinoic acid were purchasedfrom Sigma-Aldrich Sweden AB. Docetaxel was purchased from ScinoPharmTaiwan, Ltd. Ixabepilone was purchased from Chemtronica KB, Sweden.Fenretinide was synthesized according to a standard procedure (CancerResearch, 39, 1339-1346, April 1979). Taxol, Taxotere and Abraxane werepurchased from pharmacy stores and reconstituted according tomanufacturers prescribing information.

Particle size of formulations was measured by dynamic light scatteringmethod with the use of a red laser (633 nm). Zeta(Z)-potential wasmeasured by electrophoretic light scattering method. Nano-ZS (MalvernInstruments Ltd.) was used for determination both particle size andzeta-potential. Average values of three independent measurements werecalculated for plotting of particle size and zeta-potential behaviour.Y-error bars are composed by +/− standard deviation of the measurements.

For evaluation of cytotoxicity in vitro the cells of different humantumour cell lines were purchased from American Type Culture Collection(Rockville, Md., USA): Human Breast Adenocarcinoma Cell Line MDA-MB-231(ATCC-HTB-26, Lot 3576799), Human Ovary Adenocarcinoma Cell Line SKOV-3(ATCC-HTB-77, Lot 3038337) and Human Lung Non-Small Cancer Cell LineA549 (ATCC-CCL-185, Lot 3244171). MDA-MB-231 cells were propagated inMEM culture medium with 2 mM L-glutamine, 10% fetal bovine serum (FBS)and antibiotics. SKOV-3 cells were cultured in McCoy's 5A culturemedium, supplemented with 1,5 mM L-glutamine, 10% FBS and antibiotics.All media and supplements were purchased from Sigma-Aldrich Co. (St.Louis, Mo., USA). Cell propagation of all lines was carried out in BDFalcon™ 25 or 75 cm² cultivation flasks (Becton Dickinson Labware). A549cells were cultured in Ham's F-12 culture medium with 1 mM L-glutamine,10% FBS and antibiotics. Cell propagation of all lines was carried outin BD Falcon™ 25 or 75 cm² cultivation flasks.

Drug cytotoxicity testing was carried out using BD Falcon™ 96-wellcultivation plates for adherent cells (Becton Dickinson Labware). Theseplates were seeded by cells at 8×10³ cells/well for MDA-MB-231, at10×10³ cells/well for SKOV-3 or at 6×10³ cells/well for A549 in a volumeof 200 μl/well. Both flasks and cultivation plates were incubated forcell growth at 37° C. in a humidified atmosphere of 95% air and 5% CO₂.

The cell cultures in the cultivation plates were allowed to adhere for24 hour of incubation. On day 1 after cell seeding 4 μL solutions of theformulations to be tested with different concentrations in appropriatesolvents were added to wells with cultures (dose—response experiments).In the control cultures 4 μL of the solvents were added as solventcontrol. The cells were incubated within 2-4 consecutive days. At theend of the incubation period adherent cells were detached bytrypsinization and the number of viable cells was counted using trypanblue exclusion test and a hemocytometer. All experiment were performedat least three times and data were derived from an average of threedeterminations each in four replicates. The results were expressed asmean cell number±SE and the differences between control and test seriesevaluated by means of Student's t-test. The drug cytotoxicity wasevaluated based on the extent of cell growth inhibition. The cell growthinhibition by the tested drugs was calculated as follows:

${{Cell}\mspace{14mu} {growth}\mspace{14mu} {inhibition}\mspace{14mu} \%} = {\frac{{Control} - {{Test}\mspace{14mu} {Series}}}{Control} \times 100}$

In control series 4 μL of different solvents used for drug testing wereadded to cultures as negative solvent controls. The differences betweenthese control series were insignificant; therefore an average ofnegative controls was applied for calculations.

Solutions of paclitaxel and docetaxel as well as their commercialformulations were used as positive controls. The differences in growthinhibition by these drugs in different solvents were insignificant;therefore an average inhibition of positive controls was applied forcalculations.

The mean IC₅₀±SE was calculated on the bases of at least three separateexperiments.

Enhancement factors (EF) were calculated by dividing of IC₅₀ of thecontrol comparison drug with IC₅₀ of the inventive formulation.

The ionic strength of a solution is a function of the concentration ofall ions present in a solution.

$I_{c} = {\frac{1}{2}{\sum\limits_{B = 1}^{n}\; {c_{B}z_{B}^{2}}}}$

where c_(B) is the concentration of ion B, z_(B) is the charge number ofthat ion, and the sum is taken over all ions in the solution.

Example 1

Preparation of Amorphous Paclitaxel

12 ml of a paclitaxel stock solution in methanol (c=2.5 mg/ml) and 2 mlof an aqueous solution of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid (c=15 mg/ml) were evaporated in vacuoto dryness in a 50 ml round bottom flask. 15 ml of methanol was added tothe flask and the residue was dissolved. The obtained solution wasevaporated to dryness. The film obtained after the evaporation consistedof a mixture of amorphous paclitaxel and a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid.

Example 2

Preparation of Amorphous Docetaxel

27 ml of a docetaxel stock solution in methanol (c=0.5 mg/ml) and 1 mlof an aqueous solution of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid (c=15 mg/ml) were combined in a 100 mlround bottom flask. The obtained solution was evaporated in vacuo todryness; the residue was dissolved in 20 ml of methanol followed by anew evaporation of methanol in vacuo. The film obtained after theevaporation consisted of a mixture of amorphous docetaxel and a sodiumsalt of the methyl ester of N-13-cis-retinoyl cysteic acid.

Example 3

Dissolving of Amorphous Paclitaxel in Micellar Solution of a Sodium Saltof the Methyl Ester of N-All-Trans-Retinoyl Cysteic Acid

13 ml of water and 2 ml of an aqueous solution of a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid (c=15 mg/ml) wereadded to the flask containing the film with amorphous paclitaxelprepared in Example 1. The paclitaxel film was completely dissolved bygentle shaking of the vial for 10 min. The obtained solution was clearand transparent. It contained dissolved paclitaxel in a concentration of2 mg/ml. Filtration the solution through 0.2 μm filter did not result inany reduction of the paclitaxel concentration.

Example 4

Dissolving of Amorphous Docetaxel in Aqueous Solution of a Sodium Saltof the Methyl Ester of N-13-cis-retinoyl Cysteic Acid

24.4 ml of water was added to the amorphous docetaxel obtained inExample 2, and the mixture was stirred by magnetic stirrer for 5minutes. Then 2.6 ml of an aqueous solution of a sodium salt of themethyl ester of N-13-cis-retinoyl cysteic acid (c=15 mg/ml) was added tothe suspension and the mixture was stirred for 15 min. The obtainedsolution was clear and transparent. It contained dissolved docetaxel ina concentration of 0.5 mg/ml. Filtration the solution through 0.2 μmfilter did not reveal any reduction of the docetaxel concentration.

Example 5

Preparation of Paclitaxel Aqueous Formulation by the Step-Wise Mixing ofAqueous Solution of a Mixture of a Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl Cysteic Acid and a Sodium Salt of the Methyl Esterof N-13-cis-retinoyl Cysteic Acid and Methanol Solution of Paclitaxel

10 ml of a methanol solution of paclitaxel (10 mg/ml) was added dropwiseinto a 500 ml round bottom flask containing 120 ml of an aqueoussolution of a sodium salt of the methyl ester of N-all-trans-retinoylcysteic acid (2.5 mg/ml) and a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid (2.5 mg/ml) while stirring by means of amagnetic stirrer. Then the content of the flask was evaporated on arotary evaporator at 90 rpm and a bath temperature 45° C. until theinternal pressure of the closed vacuum system consisting of the flask,the evaporator and a vacuum pump dropped to 70 mbar. Such addition ofpaclitaxel methanol solution as described above followed by evaporationwas repeated twice. The total volume of added methanol solution was 30ml. The aqueous solution remaining after the evaporation was transferredfrom the flask into a 250 ml measuring cylinder. The flask was rinsedthree times with 5 ml of water and the rinsing solutions were pouredinto the cylinder. To the combined solutions was added water to achievea total volume 150 ml. The obtained solution was filtered through a 0.2μm filter and freeze dried. The paclitaxel concentration in the obtainedformulation was 2 mg/ml.

Example 6

Preparation of Docetaxel Aqueous Formulation by the Step-Wise Mixing ofAqueous Solution of a Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl Cysteic Acid and Ethanol Solution of Docetaxel

6 ml of a solution of docetaxel (5 mg/ml) in 95% ethanol was addeddropwise into a 500 ml round bottom flask containing 100 ml of anaqueous solution of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid (3 mg/ml) while stirring by means of amagnetic stirrer. The main part of the ethanol was evaporated on arotary evaporator at 90 rpm and a bath temperature 55° C. until theinternal pressure of the closed vacuum system consisting of the flask,the evaporator and a vacuum pump dropped to 60 mbar. Such addition ofdocetaxel ethanol solution as described above followed by evaporationwas repeated twice. The total volume of added ethanol solution was 30ml. The aqueous solution remaining after the evaporation of ethanol wastransferred from the flask into a 250 ml measuring cylinder. The flaskwas rinsed three times with 5 ml water and the rinsing solutions werepoured into the cylinder. Water was added to the combined solutions toachieve a total volume 150 ml. After filtration through a 0.2 μm filterthe formulation was freeze dried. The docetaxel concentration in theobtained formulation was 1 mg/ml.

Example 7

Preparation of Docetaxel Aqueous Formulation by the Mixing of AqueousSolution of a Sodium Salt of the Methyl Ester of N-all-trans-retinoylCysteic Acid and a Sodium Salt of the Methyl Ester of N-13-cis-retinovlCysteic Acid and Ethanol Solution of Docetaxel During Evaporation.

A 1000 ml round bottom flask containing 150 ml of an aqueous solution ofa sodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid(3 mg/ml) and a sodium salt of the methyl ester of N-13-cis-retinoylcysteic acid (3 mg/ml) was attached to a rotary evaporator equipped withan inlet pipe for feeding of alcohol solutions of taxanes in such a waythat the inlet pipe did not come in touch with the aqueous solution. Theevaporation started with a bath temperature of 45° C. and a rotationspeed of 100 rpm. After 1 min dropwise addition (60 drops/min or 3ml/min) of 80 ml of a methanol solution of docetaxel (5 mg/ml) wasstarted. After this addition had been completed the evaporation wascontinued for 5 min. The aqueous solution remaining after theevaporation of methanol was transferred from the evaporating flask intoa 250 ml measuring cylinder. The flask was rinsed three times with 10 mlwater and the rinsing solutions were poured into the cylinder. Water wasadded to the combined solutions was added to achieve a total volume 200ml. After filtration through a 0.2 μm filter the formulation was freezedried. The docetaxel concentration in the obtained formulation was 2mg/ml.

Example 8

Investigation of the Dependence of Particle Size on the w/w Ratio of aSodium Salt of the Methyl Ester of N-all-trans-retinoyl CysteicAcid/Paclitaxel in Formulations Formed by the Reconstitution of FreshlyEvaporated Residues of a Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl Cysteic Acid and Paclitaxel with Aqueous Solutionof Sodium Chloride in Concentration 9 mg/ml.

TABLE 1 w/w ratio of sodium salt of Paclitaxel concentration Paclitaxelconcentration Paclitaxel concentration Paclitaxel concentration methylester of 0.5 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml N-all-trans- Average AverageAverage Average retinoyl cysteic particle St. particle St. particle St.particle St. acid/paclitaxel size, nm dev. size, nm dev. size, nm dev.size, nm dev. 1.1 27.9 2.0 32.2 1.2 35.7 1.3 40.3 1.1 1.2 21.4 0.6 22.01.1 23.5 1.2 25.6 0.8 1.5 13.1 0.6 14.8 0.5 14.9 1.0 15.3 0.7 3.0 12.60.3 13.3 0.7 13.8 0.4 14.8 0.5 8.0 11.0 0.5 11.5 0.6 12.8 0.4 13.3 0.3

As shown in Table 1 and FIG. 1 the particle size decreases with thereduction of amount of paclitaxel which is loaded in micelles.

Example 9

Investigation of the Dependence of Particle Size of DocetaxelFormulation on the Concentration of Sodium Chloride.

The solutions were prepared by reconstitution of freeze-dried powdercontained docetaxel and a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid in the w/w ratio 1:1.

TABLE 2 Docetaxel concentration Docetaxel concentration Docetaxelconcentration Docetaxel concentration Concentration 0.5 mg/ml 1 mg/ml 2mg/ml 4 mg/ml of NaCl, Average St. Average St. Average St. Average St.mg/ml size, nm dev. size, nm dev. size, nm dev. size, nm dev. 4 7.2 0.76.7 0.6 6.4 0.4 5.9 2.5 8 7.8 0.7 8.2 0.7 9.3 1.4 12.7 1.4 12 12.1 1.013.4 0.9 14.6 1.0 40.0 4.9 16 17.0 2.3 29.0 4.2 51.3 3.7 82.7 3.7 2022.4 1.8 39.3 2.8 72.3 3.7 107.7 6.2 24 28.3 4.6 86.0 4.2 108.3 7.5144.3 9.9

As shown in Table 2 and FIG. 2 the increase in concentration of sodiumchloride, i.e. ionic strength, makes the particles larger.

Example 10

Transformation of a Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl Cysteic Acid into its Calcium Salt.

Aqueous solutions of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid (5 ml, 15 mg/ml) and calcium chloride(3 ml, 30 mg/ml) were mixed in a 10 ml test tube. During the mixing afine precipitation emerged. The precipitate was separated bycentrifugation of the test tube at 3000 rpm for 10 min. The supernatantwas removed and the precipitate was shaken with 8 ml of water followedby a new centrifugation. After three additional washing procedures asdescribed above the supernatant was filtered through a 0.2 μm filter inorder to remove possible large aggregates of the product. The solubilityof the calcium salt of the methyl ester of N-all-trans-retinoyl cysteicacid corresponded to its concentration in the filtered solution and wasequal to 0.2 mg/ml as measured by the UV method described above. Thereaction is illustrated by the below general scheme involving chloridesof any polyvalent metal ions, not only calcium ions.

${\underset{{{Water}{\mspace{11mu} \;}{soluble}\mspace{14mu} {sodium}}{{salt}\mspace{14mu} {of}\mspace{14mu} {methyl}\mspace{14mu} {ester}\mspace{14mu} {of}}{N - {all} - {trans} - {retinoyl}}{{cysteic}\mspace{14mu} {acid}\mspace{14mu} {or}\mspace{14mu} {methyl}}{{{ester}\mspace{14mu} {of}\mspace{14mu} N} - 13 - {cis} - {{retinoyl}\mspace{14mu} {cysteic}\mspace{14mu} {acid}}}}{n\; R\text{-}{{SO}_{3}}^{-}{Na}^{+}} + {{Metal}^{n +}{Cl}_{n}^{-}}}->{\underset{{{Water}\mspace{14mu} {insoluble}\mspace{14mu} {salt}\mspace{14mu} {of}\mspace{14mu} {methyl}\mspace{14mu} {ester}}{{{of}\mspace{14mu} N} - {all} - {trans} - {{retinoyl}\mspace{14mu} {cysteic}\mspace{14mu} {acid}}}{{{or}\mspace{14mu} {methyl}\mspace{14mu} {ester}\mspace{14mu} {of}\mspace{14mu} N} - 13 - {cis} - {retinoyl}}{{cysteic}\mspace{14mu} {acid}\mspace{14mu} {with}\mspace{14mu} {polyvalent}\mspace{14mu} {metal}}}{\left( {R\text{-}{{SO}_{3}}^{-}} \right)_{n}\left. {Metal}^{n^{+}}\downarrow \right.} + {n\; {Na}^{+}{Cl}^{-}}}$

Example 11

Investigation of the Dependence of Particle size of PaclitaxelFormulation on the Concentration of Calcium Chloride.

Solutions were prepared by reconstitution of freeze-dried powdercontaining paclitaxel, a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid and a sodium salt of the methyl esterof N-13-cis-retinoyl cysteic acid in the w/w/w ratio 1:1:1. Solvents forthe reconstitution were prepared by dissolving of appropriate amounts ofcalcium chloride dihydrate in an aqueous solution of sodium chloridewith a concentration 9 mg/ml.

TABLE 3 Paclitaxel concentration Paclitaxel concentration Paclitaxelconcentration 0.5 mg/ml 1 mg/ml 2 mg/ml Concentration Average AverageAverage of CaCl₂, particle St. particle St. particle St. mmol/l size, nmdev. size, nm dev. size, nm dev. 0 12.7 0.4 16.3 1.1 22.1 0.2 2 23.8 1.724.6 0.5 27.3 0.2 4 27.4 0.2 30.1 0.4 32.0 0.1 6 51.0 0.6 55.2 5.1 58.61.6

As shown in Table 3 and FIG. 3 the size of particles in the formulationsincreases almost linearly with the increase of CaCl₂ concentration.

Example 12

Time Course of Particle Size and Zeta-Potential of Formulation Obtainedby Reconstitution of Freeze Dried Mixture of Paclitaxel, a Sodium Saltof the Methyl Ester of N-all-trans-retinoyl Cysteic Acid and a SodiumSalt of the Methyl Ester of N-13-cis-retinoyl cysteic acid in w/w/wratio 1:0.75:0.75 in Aqueous Solution of Sodium Chloride (9 mg/ml),Calcium Chloride (2 mmol/l) and Magnesium Chloride (1 mmol/l)

TABLE 4 Paclitaxel concentration Paclitaxel concentration Paclitaxelconcentration 0.5 mg/ml 1 mg/ml 2 mg/ml Average Average Average Timeafter particle St. particle St. particle St. reconstitution size, nmdev. size, nm dev. size, nm dev. 0 22.1 0.5 23.5 0.5 25.6 0.8 1 22.7 0.724.1 0.8 26.3 0.7 2 23.1 0.5 24.3 0.4 26.2 0.5 4 23 0.4 24.4 0.3 26.60.2 8 23.4 0.7 24.0 0.6 27.0 0.4

TABLE 5 Paclitaxel concentration Paclitaxel concentration Paclitaxelconcentration 0.5 mg/ml 1 mg/ml 2 mg/ml Zeta- Zeta- Zeta- Time afterpotential, St. potential, St. potential, St. reconstitution mV dev. mVdev. mV dev. 0 −24.5 1.3 −28.7 1.2 −29.9 1.1 1 −26.3 1.8 −30.1 1.0 −32.70.8 2 −25.2 0.4 −30.4 1.0 −30.6 0.5 4 −27.0 0.5 −29.6 0.6 −31.2 0.3 8−27.1 0.4 −30.4 0.3 −32.4 0.6

Table 4 and 5, and FIGS. 4 and 5 show that there are no any significantchanges in the values of the particle size as well as Zeta-potentialduring storage of the formulation for 8 hours.

Example 13

Time Course of Particle Size and Zeta-potential of Formulation Obtainedby Reconstitution of Freeze Dried Mixture of Docetaxel and a Sodium Saltof the Methyl Ester of N-all-trans-retinoyl Cysteic Acid in w/w ratio1:2 in Aqueous Solution of Sodium Chloride (9 mg/ml) and CalciumChloride (3 mmol/l)

TABLE 6 Docetaxel concentration Docetaxel concentration Docetaxelconcentration 0.5 mg/ml 1 mg/ml 2 mg/ml Average Average Average Timeafter particle St. particle St. particle St. reconstitution size, nmdev. size, nm dev. size, nm dev. 0 11.9 0.3 12.6 0.2 13.1 0.4 1 12.3 0.313.2 0.4 13.4 0.2 2 12.4 0.2 13.0 0.2 13.7 0.4 4 12.2 0.4 12.9 0.1 13.40.2 8 12.5 0.3 13.2 0.2 13.8 0.2

TABLE 7 Paclitaxel concentration Paclitaxel concentration Paclitaxelconcentration 0.5 mg/ml 1 mg/ml 2 mg/ml Zeta- Zeta- Zeta- Time afterpotential, St. potential, St. potential, St. reconstitution mV dev. mVdev. mV dev. 0 −22.2 2.1 −22.6 1.3 −22.8 0.6 1 −23.4 0.9 −22.4 1.2 −24.10.8 2 −22.7 0.4 −23.7 0.9 −23.3 0.4 4 −21.9 0.3 −23.1 0.8 −23.1 0.2 8−21.7 0.6 −23.4 0.6 −23.5 0.5

Table 6 and 7, and FIGS. 6 and 7 show that there are no any significantchanges in the values of the particle size as well as Zeta-potentialduring storage of the formulation for 8 hours.

Example 14

Preparation of Amorphous Ciclosporin A

50 ml of a Ciclosporin A stock solution in methanol (c=1.0 mg/ml) and4.2 ml of an aqueous solution of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid (c=12 mg/ml) were evaporated in vacuoto dryness in a 100 ml round bottom flask. 15 ml of methanol was addedto the flask and the residue was dissolved. The obtained solution wasevaporated to dryness. The film obtained after the evaporation consistedof a mixture of amorphous Ciclosporin A and a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid.

Example 15

Dissolving of Amorphous Ciclosporin A in Micellar Solution of a SodiumSalt of the Methyl Ester of N-All-Trans-Retinoyl Cysteic Acid

45.8 ml of water and 4.2 ml of an aqueous solution of a sodium salt ofthe methyl ester of N-all-trans-retinoyl cysteic acid (c=12 mg/ml) wereadded to the flask containing the film with amorphous Ciclosporin Aprepared in Example No 14. The Ciclosporin A film was completelydissolved by gentle shaking of the vial for 10 min. The obtainedsolution was clear and transparent. It contained dissolved Ciclosporin Ain a concentration of 1 mg/ml. Filtration the solution through 0.2 μmfilter did not result in any reduction of the Ciclosporin Aconcentration.

Example 16

Investigation of the Dependence of Particle Size on the w/w Ratio of aSodium Salt of the Methyl Ester of N-all-trans-retinoyl cysteicacid/Ciclosporin A in Formulations Formed by the Reconstitution ofFreshly Evaporated Residues of a Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl cysteic acid and Ciclosporin A with AqueousSolution of Sodium Chloride in Concentration 9 mg/ml.

TABLE 8 w/w ratio of sodium salt of methyl ester Ciclosporin Aconcentration Ciclosporin A concentration Ciclosporin A concentrationCiclosporin A concentration of N-all-trans- 0.5 mg/ml 1 mg/ml 2 mg/ml 4mg/ml retinoyl Average Average Average Average cysteic acid/ particleSt. particle St. particle St. particle St. Ciclosporin A size, nm dev.size, nm dev. size, nm dev. size, nm dev. 1.3 57.3 1.6 63.2 2.2 69.7 2.078.0 3.6 1.4 42.2 1.8 46.9 1.8 50.8 1.8 58.8 2.7 1.6 28.6 1.4 30.9 1.732.3 1.1 37.8 2.5 2.0 20.2 1.2 22.1 1.1 25.5 0.6 25.9 0.9 8.0 10.4 0.811.5 0.4 11.9 0.5 12.9 0.5

As shown in Table 8 and FIG. 10 the particle size decreases with thereduction of amount of Ciclosporin A which is loaded in micelles.

Biological Evaluation

Examples 17-21

In vitro experiments showed that the activity of taxane formulations indifferent solid tumour cell lines is more expressed by the use ofnanoparticles as provided by the present invention. Moreover thecytotoxicity of these formulations dramatically depends on the size ofthe nanoparticles. Bigger size of the nanoparticles in the inventivedrug delivery system leads to diminished transport of taxanes in a cell,which in turn results in reduction of the cytotoxicity.

The highest activity was observed when the size was 25 and 13 nm forpaclitaxel and docetaxel solutions, respectively: in vitro experimentsgave enhancement factors for these formulations of 41.7 and 31.7,respectively, on day 3 of exposure. The control sample in thisexperiment contained taxanes in ethanol solutions (without anynanoparticles).

Other in vitro comparisons of taxane formulations according to theinvention with commercially available taxane formulations showed thatthe formulations according to the invention possess more expressedcytotoxic activity against different malignant cell culture lines likebreast adenocarcinoma, ovary adenocarcinoma and lung non-small cellcancer.

Example 17

Comparative Evaluation of the Cytotoxicity of the Formulations Formed byDocetaxel-Sodium Salt of the Methyl Ester of N-all-trans-retinoylCysteic Acid-Sodium Salt of the Methyl Ester of N-13-cis-retinoylCysteic Acid Mixture (w/w/w=1:1:1) in Cultures of Human OvaryAdenocarcinoma SKOV3 Cell Line

Freeze dried powder consisted of docetaxel, a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid and a sodium salt of themethyl ester of N-13-cis-retnoyl cysteic acid was dissolved in either70% ethanol or sodium chloride solution (9 mg/ml) containing anappropriate amount of calcium chloride. Samples of the solutionsobtained were taken and used for measurement of average particle size.

TABLE 9 Concentration Particle of CaCl₂, size, Day 3 EF* Day 4 EF* Day 5EF* Solvent mmol/l nm IC₅₀ day 3 IC₅₀ day 4 IC₅₀ day 5 70% EtOH — — 2.0· 10⁻⁷ — 7.2 · 10⁻⁹ — 7.6 · 10⁻¹⁰ — NaCl solution 0 11.3 1.2 · 10⁻⁷ 1.77.2 · 10⁻⁹ 1 6.5 · 10⁻¹⁰ 1.2 NaCl solution 1 12.2 3.4 · 10⁻⁸ 5.9 5.6 ·10⁻⁹ 1.3 4.2 · 10⁻¹⁰ 1.8 NaCl solution 2 13.1 6.3 · 10⁻⁹ 31.7 2.1 · 10⁻⁹3.4 9.4 · 10⁻¹¹ 8.1 NaCl solution 3 14.6 2.0 · 10⁻⁸ 10 3.4 · 10⁻⁹ 2.11.4 · 10⁻¹⁰ 5.4 *The formulation with ethanol was used as positivecontrol for calculation of EF

Table 9 and FIG. 8 show that the formulation contained 2 mmol/l ofcalcium is the most active. Then with increase and decrease of calciumconcentration the cytotoxicity of the formulations is reduced.

Example 18

Comparative Evaluation of the Cytotoxicity of the Formulations Formed byPaclitaxel-Sodium Salt of the Methyl Ester of N-all-trans-retinoylCysteic Acid-Sodium Salt of the Methyl Ester N-13-cis-retinoyl CysteicAcid Mixture (w/w/w=1:0.75:0.75) in Cultures of Human OvaryAdenocarcinoma SKOV3 Cell Line

Freeze dried powder consisting of paclitaxel, a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid and a sodium salt ofthe methyl ester of N-13-cis-retinoyl cysteic acid was dissolved ineither 70% ethanol or sodium chloride solution (9 mg/ml) containing anappropriate amount of calcium chloride. Samples of the solutionsobtained were taken and used for measurement of average particle size.

TABLE 10 Concentration Particle of CaCl₂, size, Day 3 EF* Day 4 EF* Day5 EF* Solvent mmol/l nm IC₅₀ day 3 IC₅₀ day 4 IC₅₀ day 5 70% EtOH — —5.0 · 10⁻⁶ — 2.1 · 10⁻⁷ — 6.8 · 10⁻⁸ — NaCl solution 0 17 3.0 · 10⁻⁶ 1.71.3 · 10⁻⁷ 1.6 5.5 · 10⁻⁸ 1.2 NaCl solution 1 19 1.7 · 10⁻⁶ 2.9 8.4 ·10⁻⁸ 2.5 9.8 · 10⁻⁹ 6.9 NaCl solution 2 25 1.2 · 10⁻⁷ 41.7 2.3 · 10⁻⁸9.1  7.2 · 10⁻¹⁰ 94.0 NaCl solution 3 29 2.4 · 10⁻⁷ 20.8 4.3 · 10⁻⁸ 4.91.2 · 10⁻⁸ 5.7 *The formulation with ethanol was used as positivecontrol for calculation of EF

Table 10 and FIG. 9 show that the formulation contained 2 mmol/l ofcalcium is the most active. Then with increase and decrease of calciumconcentration the cytotoxicity of the formulations is reduced.

Example 19

Evaluation of Cytotoxicity of the Formulation “Paclitaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.75:075)” withTAXOL®, ABRAXANE® and Paclitaxel alone in Cultures of Human BreastAdenocarcinoma MDA-MB-231 Cell Line.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Paclitaxel was used in a methanol solution. TAXOL®and ABRAXANE® samples were prepared according to instructions from themanufacturers by dilution of a commercially available TAXOL® concentrate(6 mg/ml) in sodium chloride (9 mg/ml) solution and by reconstitution offreeze dried albumin-bound paclitaxel with sodium chloride (9 mg/ml)solution to a paclitaxel concentration of 5 mg/ml. All samples were usedwithin one hour after preparation. Enhancement effects were calculatedversus paclitaxel methanol solution. The results are set forth in Table11 below.

TABLE 11 Particle EF EF Formulation size, nm IC₅₀ day 3 day 3 IC₅₀ day4day 4 Paclitaxel — (3.80 ± 0.15) × — (3.4 ± 0.12) × — 10⁻⁸ 10⁻⁸ TAXOL ®— (2.04 ± 0.05) × 1.9 (2.0 ± 0.10) × 1.7 10⁻⁸ 10⁻⁸ ABRAXANE ® 130 (4.2 ±0.09) × 0.9 (3.4 ± 0.16) × 1 10⁻⁸ 10⁻⁸ Paclitaxel-sodium  24 (4.2 ±0.14) × 4.9 (3.2 ± 0.09) × 10.6 salt of methyl ester 10⁻⁹ 10⁻⁹ ofN-all-trans- retinoyl cysteic acid- sodium salt of methyl ester ofN-13-cis- retinoyl cysteic acid

Example 20

Evaluation of Cytotoxicity of the Formulation “Docetaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.5:0.5)” withTAXOTERE® and Docetaxel alone in Cultures of Human Breast AdenocarcinomaMDA-MB-231 Cell Line.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Docetaxel was used in a methanol solution.TAXOTERE® sample was prepared according to instructions from themanufacturer by dilution of a commercially available concentrate (40mg/ml) firstly with ethanol solution to concentration 10 mg/ml followedby further dilution in sodium chloride (9 mg/ml) solution. All sampleswere used within one hour after preparation. Enhancement effects werecalculated versus docetaxel methanol solution. The results are set forthin Table 12 below.

TABLE 12 Particle EF EF size, day day Formulation nm IC₅₀ day 3 3 IC₅₀day4 4 Docetaxel — (1.25 ± 0.11) × — (1.0 ± 0.1) × — 10⁻⁸ 10⁻⁸TAXOTERE ® — (1.08 ± 0.09) × 1.2 (9.60 ± 0.18) × 1.0 10⁻⁸ 10⁻⁹Docetaxel-sodium 12 (3.1 ± 0.1) × 4.0 (1.2 ± 0.1) × 8.3 salt of 10⁻⁹10⁻⁹ methyl ester of N-all-trans- retinoyl cysteic acid-sodium salt ofmethyl ester of N-13- cis-retinoyl cysteic acid

Example 21

Evaluation of Cytotoxicity of the Formulation “Paclitaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.75:075)” withTAXOL®, ABRAXANE® and Paclitaxel alone in Cultures of Human OvaryAdenocarcinoma SKOV-3 Cell Line.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Paclitaxel was used in a methanol solution. TAXOL®and ABRAXANE® samples were prepared according to instructions from themanufacturers by dilution of a commercially available TAXOL® concentrate(6 mg/ml) in sodium chloride (9 mg/ml) solution and by reconstitution offreeze dried albumin-bound paclitaxel with sodium chloride (9 mg/ml)solution to a paclitaxel concentration of 5 mg/ml. All samples were usedwithin one hour after preparation. Enhancement effects were calculatedversus paclitaxel methanol solution. The results are set forth in Table13 below.

TABLE 13 Particle EF EF Formulation size, nm IC₅₀ day 3 day 3 IC₅₀ day4day 4 Paclitaxel — (5.94 ± — (6.22 ± — 0.21) × 10⁻⁷ 0.18) × 10⁻⁸ TAXOL ®— (3.24 ± 1.8 (4.20 ± 1.5 0.16) × 10⁻⁷ 0.25) × 10⁻⁸ ABRAXANE ® 130 (6.3± 0.94 (6.5 ± 0.96 0.32) × 10⁻⁷ 0.30) × 10⁻⁸ Paclitaxel-sodium  24 (2.05± 2.3 (2.17 ± 2.9 salt of methyl 0.08) × 10⁻⁷ 0.15) × 10⁻⁸ ester ofN-all- trans- retinoyl cysteic acid- sodium salt of methyl ester ofN-13- cis-retinoyl cysteic acid

Example 22

Evaluation of Cytotoxicity of the Formulation “Docetaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.5:0.5)” withTAXOTERE® and Docetaxel alone in Cultures of Human Ovary AdenocarcinomaSKOV-3 Cell Line.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Docetaxel was used in a methanol solution.TAXOTERE® sample was prepared according to instructions from themanufacturer by dilution of a commercially available concentrate (40mg/ml) firstly with ethanol solution to concentration 10 mg/ml followedby further dilution in sodium chloride (9 mg/ml) solution. All sampleswere used within one hour after preparation. Enhancement effects werecalculated versus docetaxel methanol solution. The results are set forthin Table 14 below.

TABLE 14 Particle EF EF Formulation size, nm IC₅₀ day 3 day 3 IC₅₀ day4day 4 Docetaxel — (9.07 ± — (2.85 ± — 0.38) × 10⁻⁸ 0.26) × 10⁻⁸TAXOTERE ® — (1.18 ± 0.8 (2.03 ±  1.4 0.09) × 10⁻⁷ 0.15) × 10⁻⁹Docetaxel-sodium 12 (3.24 ± 2.8 (2.86 ± 10.0 salt of 0.18) × 10⁻⁹ 0.13)× 10⁻⁹ methyl ester of N-all- trans-retinoyl cysteic acid-sodium salt ofmethyl ester of N-13-cis- retinoyl cysteic acid

Example 23

Evaluation of Cytotoxicity of the Formulation “Paclitaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.75:075)” withTAXOL®, ABRAXANE® and Paclitaxel alone in Cultures of Human LungNon-Small Cancer Cell Line A549.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Paclitaxel was used in a methanol solution. TAXOL®and ABRAXANE® samples were prepared according to instructions from themanufacturers by dilution of a commercially available TAXOL® concentrate(6 mg/ml) in sodium chloride (9 mg/ml) solution and by a reconstitutionof freeze dried albumin-bound paclitaxel with sodium chloride (9 mg/ml)solution to a paclitaxel concentration of 5 mg/ml. All samples were usedwithin one hour after preparation. Enhancement effects were calculatedversus paclitaxel methanol solution. The results are set forth in Table15 below.

TABLE 15 Particle EF EF Formulation size, nm IC₅₀ day 3 day 3 IC₅₀ day4day 4 Paclitaxel — (8.02 ± — (5.28 ± — 0.11) × 10⁻⁹ 0.13) × 10⁻⁹ TAXOL ®— (6.49 ± 1.2  (3.77 ± 1.4 0.08) × 10⁻⁹ 0.09) × 10⁻⁹ ABRAXANE ® 130 (1.2± 0.67 (5.2 ± 1 0.06) × 10⁻⁹ 0.15) × 10⁻⁹ Paclitaxel-sodium  24 (1.61 ±5.0  (7.02 ± 7.5 salt of 0.11) × 10⁻⁹ 0.12) × 10⁻¹⁰ methyl ester ofN-all-trans- retinoyl cysteic acid- sodium salt of methyl ester ofN-13-cis- retinoyl cysteic acid

Example 24

Evaluation of Cytotoxicity of the Formulation “Docetaxel-Sodium Salt ofthe Methyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.5:0.5)” withTAXOTERE® and Docetaxel alone in Cultures of Human Lung Non-Small CancerCell Line A549.

The title formulation was prepared by dissolving freeze dried powder inan aqueous solution containing sodium chloride (6 mg/ml), potassiumchloride (0.3 mg/ml), calcium chloride hexahydrate (0.4 mg/ml), sodiumlactate (3.1 mg/ml). Docetaxel was used in a methanol solution.TAXOTERE® sample was prepared according to instructions from themanufacturer by dilution of a commercially available concentrate (40mg/ml) firstly with ethanol solution to concentration 10 mg/ml followedby further dilution in sodium chloride (9 mg/ml) solution. All sampleswere used within one hour after preparation. Enhancement effects werecalculated versus docetaxel methanol solution. The results are set forthin Table 16 below.

TABLE 16 Particle EF EF Formulation size, nm IC₅₀ day 3 day 3 IC₅₀ day4day 4 Docetaxel — (5.76 ± — (4.97 ± — 0.26) × 10⁻⁹ 0.27) × 10⁻⁹TAXOTERE ® — (4.81 ± 1.2 (4.63 ± 1.1 0.34) × 10⁻⁹ 0.17) × 10⁻⁹Docetaxel-sodium 12 (9.14 ± 6.3 (5.35 ± 7.9 salt 0.47) × 10⁻¹⁰ 0.15) ×10⁻¹⁰ of methyl ester of N- all-trans- retinoyl cysteic acid- sodiumsalt of methyl ester of N-13-cis- retinoyl cysteic acid

Example 25

A One Month Toxicity Study of Formulation “Paclitaxel-Sodium Salt of theMethyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinovl Cysteic Acid (w/w/w 1:0.75:075)” inRats

The tested formulation was prepared by reconstitution in saline offreeze dried mixture of Paclitaxel -Sodium Salt of the Methyl Ester ofN-all-trans-retinoyl Cysteic Acid-Sodium Salt of the Methyl Ester ofN-13-cis-retinoyl Cysteic Acid (w/w/w 1:0.75:075). 80 Wistar rats(BRLHan:Wist@Mol (GALAS)), 40 males and 40 females, were divided into 4groups, each of 10 males and 10 females. Tested formulations wereadministered by intravenous injection once weekly over 5 weeks. Group 1received saline and acted as controls, Group 2 received 5 mg/kg offormulation of paclitaxel with polyoxyethylated castor oil (Taxol®),Group 3 received 5 mg/kg of the title formulation, and Group 4 received10 mg/kg of the title formulation. Originally, the study was designed sothat Group 2 would receive 10 mg/kg Taxol® as a direct comparison withGroup 4, however, due to mortality, this dosage was reduced to 5 mg/kgsuch that a direct comparison with Group 3 was more appropriate. Therewere 8 deaths during the study. Seven rats received 10 mg/kg Taxol® diedshortly after their first dose. Five of these rats were replaced withspares, and the dosage was reduced to 5 mg/kg. For females in Groups 2,3 and 4 mean values for the red blood cell parameters (Hb, RBC and HT)were lower than for the controls. Although a similar change was not seenin the males, values for the red cell indices MCV in males of Group 2were elevated. Mean values for white blood cells, particularlyneutrophils, lymphocytes, eosinophils and in the males, monocytes intreated animals were lower than for controls. Mean serum bilirubinvalues for males in Group 4 and females in Group 2 and 4 were higherthan for the controls. Bilirubin for females in Group 2 (Taxol®) wassignificantly higher than for the females in Group 3. Liver weight inmales of Groups 2 and 4 was significantly lower than for the controls.Thymus weight for males and females in Group 4 and for the males inGroup 2 was significantly lower than for the control. Relatively highincidence of minimal to slight lymphoid atrophy was recorded in thespleen, the mesenteric- and mandibular lymph node of Group 4. Lowincidence of minimal to slight lymphoid atrophy was recorded in thespleen of Groups 2 and 3. The incidence lymphoid atrophy of the spleenwas slightly higher in the Group 2 males. Low incidence of minimal toslight lymphoid atrophy was rerecorded in the mesenteric- and mandibularlymph nodes of Group 2. Minimal to slight increased corticallymphocytolysis was recorded in all males of Group 2. In the mammarygland of the males from Groups 2 and 4, higher incidence of minimalmultifocal decreased secretory vacuoles/hypoplasia of alveoli wasrecorded compared to control and Group 3. Increased incidence of mitoticfigures/apoptotic bodies in the epithelial lining of the mammary glandwas recorded in approximately half of the males of all treated groups.

This example demonstrates that nano-particle formulation“Paclitaxel-Sodium Salt of the Methyl Ester of N-all-trans-retinoylCysteic Acid-Sodium Salt of the Methyl Ester of N-13-cis-retinoylCysteic Acid (w/w/w 1:0.75:075)” has a lower toxicity as compared toidentical concentrations of conventional formulation of paclitaxel withpolyoxyethylated castor oil.

Example 26

Advantages of nano-particle formulation “Paclitaxel-Sodium Salt of theMethyl Ester of N-all-trans-retinoyl Cysteic Acid-Sodium Salt of theMethyl Ester of N-13-cis-retinoyl Cysteic Acid” as compared toconventional formulation of paclitaxel with polyoxyethylated castor oil(Taxol®). The main results and conclusions are summarized in the table17 below.

TABLE 17 Comparison of paclitaxel formulations (results and set-up forthe title formulation according to a study of treatment of 34 patientswith histologically proven solid malignant tumour disease, for which nostandard therapy was available or had failed; information about Taxol ®in according to BMS PI Rev July 2007) Paclitaxel-Sodium Salt of theMethyl Ester of N-all-trans- retinoyl Cysteic Paclitaxel- Acid-SodiumSalt of polyoxy- the Methyl Ester ethylated of N-13-cis-retinoyl castorCysteic Acid oil Dose level/m² 250 175 Premedication with None Yessteroids, antiemetics and antihistamines Anaphylaxis and None 5% (Allpatients severe hypersensitivity (without received reactionspremedication) premedication) Infusion time 1 hour 3 hours

Although the invention has been described with regard to certainembodiments, including the best mode presently known to the inventors,it should be understood that various changes and modifications as wouldbe obvious to one having the ordinary skill in this art may be madewithout departing from the scope of the invention as set forth in theclaims appended hereto.

1. A drug delivery system for administration of at least onepharmaceutically active substance having a solubility per se in water ofless than about 100 μg/ml, said substance being in particulate form withan effective average particle size of less than about 50 nm, wherein thesubstance particles are essentially amorphous; the substance particlesare entrapped in nanoparticles formed of a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid, sodium salt of methyl esterof N-13-cis-retinoyl cysteic acid, or a combination thereof; theweight-to-weight ratio of said sodium salt of methyl ester ofN-all-trans-retinoyl cysteic acid, sodium salt of methyl ester ofN-13-cis-retinoyl cysteic acid, or a combination thereof, to saidsubstance is in the range from about 0.5:1 to about 20:1; and saidsubstance is a cytotoxic or a cytostatic compound.
 2. A drug deliverysystem according to claim 1, wherein the weight-to-weight ratio of saidsodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid,sodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid, or acombination thereof, to said substance is in the range from about 1:1 toabout 10:1.
 3. A drug delivery system according to claim 1, wherein saidsubstance is a taxane.
 4. A drug delivery system according to claim 1,wherein said substance is chosen from among paclitaxel, docetaxel, andderivatives thereof.
 5. A drug delivery system according to claim 1,wherein wherein said drug delivery system is for use in treatment ofcancer.
 6. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the drug delivery system according to claim
 1. 7.A pharmaceutical composition according to claim 6 in the form of anaqueous solution, a gel, a cream, an ointment, a tablet, a capsule, or asoftgel.
 8. A method for the preparation of a drug delivery systemcomprising nanoparticles formed of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or a combination thereof, and at leastone pharmaceutically active substance having a solubility per se inwater of less than about 100 μg/ml wherein said substance is provided inthe form of essentially amorphous particles with an effective averageparticle size of less than about 100 nm; the size of said nanoparticlesis controlled to have an effective average particle size of less thanabout 100 nm by adjusting the weight-to-weight ratio of said sodium saltof the methyl ester of N-all-trans-retinoyl cysteic acid, sodium salt ofthe methyl ester of N-13-cis-retinoyl cysteic acid, or combinationthereof, to said substance to be in the range from about 0.5:1 to about20:1.
 9. A method for controlling the particle size and/or particleshape and/or particle size distribution of nanoparticles formed of asodium salt of the methyl ester of N-all-trans-retinoyl cysteic acid, asodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid, or acombination thereof, and at least one pharmaceutically active substancehaving a solubility per se in water of less than about 100 pg/ml in aprocess for the preparation of a drug delivery system, wherein saidsubstance is provided in the form of essentially amorphous particleswith an effective average particle size of less than about 100 nm; theparticle size and/or particle shape and/or particle size distribution ofsaid nanoparticles is controlled by adjusting the weight-to-weight ratioof said sodium salt of the methyl ester of N-all-trans-retinoyl cysteicacid, sodium salt of the methyl ester of N-13-cis-retinoyl cysteic acid,or combination thereof, to said substance to be in the range from about0.5:1 to about 20:1.
 10. A method for controlling the particle size ofnanoparticles formed of a sodium salt of the methyl ester ofN-all-trans-retinoyl cysteic acid, a sodium salt of the methyl ester ofN-13-cis-retinoyl cysteic acid, or a combination thereof, and at leastone pharmaceutically active substance having a solubility per se inwater of less than about 100 μg/ml in a process for the preparation of adrug delivery system, wherein said substance is provided in the form ofessentially amorphous particles with an effective average particle sizeof less than about 100 nm; said essentially amorphous particles aresubmitted into and/or produced in an aqueous solution containing atleast one ionized salt, said aqueous solution having an ionic strengthI; the particle size of the nanoparticles is increased by increasing Ior decreased by decreasing I.
 11. A method for increasing the drugloading capacity of nanoparticles formed of a sodium salt of the methylester of N-all-trans-retinoyl cysteic acid, a sodium salt of the methylester of N-13-cis-retinoyl cysteic acid, or a combination thereof, andat least one pharmaceutically active substance having a solubility perse in water of less than about 100 pg/ml in a process for thepreparation of a drug delivery system by providing said substance in theform of essentially amorphous particles with an effective averageparticle size of less than about 100 nm; adjusting the weight-to-weightratio of said sodium salt of the methyl ester of N-all-trans-retinoylcysteic acid, sodium salt of methyl ester of N-13-cis-retinoyl cysteicacid, or combination thereof, to said substance to be in the range fromabout 0.5:1 to about 20:1.
 12. A method according to claim 8, whereinsaid substance is provided in the form of essentially amorphousparticles with an effective average particle size of less than about 100nm, which method comprises the steps of: dissolving said substance in asuitable organic solvent to provide an organic solution of saidsubstance; adding about 0.01-3 molar equivalents of a sodium salt of themethyl ester of N-all-trans-retinoyl cysteic acid, a sodium salt of themethyl ester of N-13-cis-retinoyl cysteic acid, or a combinationthereof, to said organic solution; and evaporating said organic solventfrom said organic solution to provide a residue which comprises thepharmaceutically active substance in the form of essentially amorphousparticles.
 13. A drug delivery system obtainable by the method accordingto claim
 8. 14. A pharmaceutical composition comprising apharmaceutically acceptable carrier and the drug delivery systemobtainable by the method according to claim
 8. 15. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thedrug delivery system obtainable by the method according to claim 8 inthe form of an aqueous solution, a gel, a cream, an ointment, a tablet,a capsule, or a softgel.
 16. A method for the treatment of cancer,wherein a pharmaceutical composition according to claim 6 isadministered in a therapeutically effective amount to a patient in needof such treatment.
 17. A method for the treatment of cancer, wherein thedrug delivery system according to claim 1 is administered in atherapeutically effective amount to a patient in need of such treatment.